Effective vaccination against HIV remains the only viable means to stop the spread of the AIDS pandemic. However, numerous attempts to elicit protective immunity to HIV have been unsuccessful. Prior efforts to elicit protective immunity have at best achieved protection in animals to the same strain that is inoculated.
A wide variety of monoclonal antibodies have been isolated from the serum of HIV infected human subjects, and these antibodies display a spectrum of activities from (a) binding just one strain of HIV-1, but not neutralizing it, to (b) binding more than one strain of HIV-1 but not neutralizing them, to (c) neutralizing just one strain of HIV-1 and either binding only that strain or binding many strains, to (d) neutralizing many strains of HIV-1 (a “broadly neutralizing” antibody). These activities suggest that these antibodies, should they be present in the serum of a human who has never been infected with the HIV virus, would protect that human from infection with the HIV-1 virus should the human be exposed to the HIV-1 virus.
The majority of antibodies generated against the HIV envelope glycoprotein gp120 are not neutralizing, either because their binding does not prevent fusion of HIV to its target cells or because the epitopes they recognize are inaccessible to the antibody. Therefore, focusing the immune response to regions of gp120 that are known to bind neutralizing antibodies may improve the efficacy of vaccination. With few exceptions, even antibodies with neutralizing activity are only reactive against a limited number of HIV strains, a result of most antibody epitopes being subject to high sequence variability.
The V3 region of the surface envelope glycoprotein of HIV-1 (120), which is the primary vaccine target in HIV-1, has given rise to many of the monoclonal antibodies in all four binding and neutralization categories that have been isolated. The V3 region of gp120, while generally variable, possesses conserved features that allow broad neutralization by certain antibodies such as the human monoclonal antibody (mAb) 447-52D (also referred to herein as 447-52 and 447). 447-52D recognizes the conserved tip of the V3 loop in a β-turn conformation.
However, most anti-V3 antibodies have narrow neutralization profiles. A specially designed V3-based immunogen that could induce high titers of antibodies with a binding mode and epitope specificity that is similar to that of one or more known broadly neutralizing antibodies (for example, 447-52D) would be expected to be valuable as an HIV vaccine.
Cholera Toxin subunit B (“CTB”) and a family of closely related bacterial proteins, such as E. coli enterotoxins, are homopentamers made of relatively small subunits (˜100 aa). The protein is highly immunogenic and has been used generally in fusion constructs to enhance immunogenicity of its fusion partner polypeptide or peptide. CTB has been described as a mucosal adjuvant for vaccines, genetically fused the ctxB gene to the psaA gene from Streptococcus pneumoniae, a surface protein, a vaccine antigen candidate. Purified CTB-PsaA expressed in E. coli, was used for intranasal immunization of mice and induced systemic and mucosal antibodies in serum, saliva, and in nasal and bronchial wash samples.
An important factor for the immunogenic property of CTB and related toxins is their binding to GM1 ganglioside. X-ray structures of CTB revealed that the oligosaccharide binding sites are formed by residues E51, Q56, H57, Q61, W88, N90, and K91. The availability of this structural information allows protein design that avoids or minimizes disruption of the CTB GM1 binding site, thereby preserving the inherent immunogenicity of these polypeptides. When used as a delivery means of a vaccine to mucosal immune systems, CTB cannot tolerate large-protein fusion which impairs pentamerization and lowers affinity for GM1-ganglioside. A new strategy to reduce steric hindrance between CTB-antigen fusion subunits promoted integration of unfused CTB “molecular buffers” into the pentamer unit, leading to more efficient self-assembly into biologically active pentamers. The chimeric protein took on a compact configuration, becoming small enough to be secreted. Affinity-purified proteins administered by a mucosal route induced specific immune responses in mice, a finding that was considered broadly applicable to bacterial enterotoxin-based vaccine design.
Its propensity to induce mucosal immunity is another advantage of CTB as an immunogenic “carrier” that is uncommon, yet is highly desirable for an HIV immunogen or vaccine, because infection commonly occurs via a mucosal route. Furthermore, CTB is not toxic without the concomitant presence of the A subunit (that is part of the native cholera toxin). CTB has been approved as a component of an anti-cholera vaccine for use in humans.
In an attempt to generate an immunogen competent to generate 447-52D-like antibodies, the known epitope of 447-52D was inserted at three different surface loop locations in the small, stable protein Escherichia coli thioredoxin (Trx). At one of the three locations (between residues 74 and 75), the insertion was tolerated (i.e., the resulting protein was stable and soluble) and bound 447-52D with an affinity similar to that of intact gp120. Upon immunization, with the V3 peptide-Trx scaffold, anti-V3 antibodies were induced that could compete for 447-52D binding to gp120. These anti-V3 antibodies were said to recognize the same epitope as 447-52D. The 447-52D-lik Abs were estimated to be present at concentrations of 50400 μg/ml of serum and were unable to effect neutralization of HIV-1 strains like JR-FL and BAL but could neutralize the sensitive MN strain. The authors suggested that because of the low accessibility of the V3 loop on primary HIV-1 isolates such as JRFL, it will be difficult to elicit a V3-specific, 447-52D-like antibody response to effectively neutralize such isolates.
It has also been observed that if the HIV-MN V3 epitope is placed in a scaffold, only strain-specific neutralization (of the MN strain) occurs.
An important surrogate end point for using the information represented by these monoclonal antibodies to eventually elicit protective immunity in humans to HIV is the capability to specifically elicit an animal serum antibody response that mimics the behavior of a specific monoclonal antibody. This capability is equally important in general for specifically utilizing similar functionally promising monoclonal antibodies to elicit protective immunity to any infectious human pathogen. A specially designed V3-based immunogen that could induce high titers of antibodies with a binding mode and epitope specificity that is similar to that of one or more known broadly neutralizing antibodies (for example, 3074) is expected to be valuable as an HIV vaccine.
Amino-acid sequence pattern in proteins that have biological significance are termed sequence motifs and usually are a low resolution representation of a stereotypical three-dimensional structural shape in the protein that is of functional or biological significance. Therefore, neutralization epitopes targeted by antibodies, which are stereotypical three-dimensional structural shapes on the molecular surface of pathogenic proteins, can be described by sequence motifs.
However, sequence motifs would not have been utilized by skilled scientists for the purposes of antigen design. First, it would not have been expected that a method to design an antigen presenting a particular epitope by way of one-dimensional sequence motifs would be superior to methods aiming to copy the three-dimensional shape of the epitope. Second, deleting epitopes by point mutations while preserving one desired epitope would not have been thought to be effective because numerous epitopes are always present and which antibodies do and do not arise from a particular protein despite the presence of many epitopes is not known. Third, whether motifs for antibody binding epitopes or motifs for antibody neutralization epitopes are more effective at eliciting specific near-monoclonal antibody responses in serum is not known. Not surprisingly then, the use of sequence motifs for epitopes as immunogen design tools, and as tools to analyze antibody responses in serum resulting from immunogen inoculation in animals and humans has never been described.
The present invention is directed to overcoming these deficiencies in the art.